Abstract: Objective: To elucidate the interaction between prostate cancer (PCa) cells and cancer-associated fibroblasts (CAFs) mediated by exosomal metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), and to explore the role and mechanism of CAFs-derived exosomal MALAT1 in the glucose metabolic reprogramming of PCa. Methods: The Cancer Genome Atlas (TCGA) data were analyzed to explore the expression pattern of MALAT1 in a variety of tumors, especially its clinical association with PCa. Single-cell RNA sequencing (scRNA-seq) and related technologies were used to investigate the mechanism of MALAT1 in mediating the interaction between PCa cells and CAFs. MALAT1 was overexpressed in CAFs cells, supernatant exosomes were extracted and co-cultured with PCa cells, and the function and mechanism of action of exosomal MALAT1 in the progression of PCa were investigated by comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) and other methods. Results: Using the TCGA dataset, it was observed that MALAT1 was associated with the malignancy degree of various tumors, including prostate adenocarcinoma, breast invasive carcinoma and bile duct carcinoma, as well as with the poor prognosis of PCa. The scRNA-seq analysis showed that not only did KLK3, a molecular marker of PCa, exist in the CAFs in the PCa tumor microenvironment, but also the expression of MALAT1 was significantly higher in CAFs than in PCa cells, suggesting that PCa cells were able to“domesticate”CAFs and promote PCa progression through the MALAT1 in CAFs. Furthermore, co-culture and ChIRP-MS experiments revealed that exosomal MALAT1 regulated the energy metabolism of PCa through direct binding to key enzymes of PCa glucose metabolism, and the combined analysis of ChIRP-MS and RNA-seq revealed that exosomal MALAT1 could directly bind to four key glycolytic genes (GPI, ALDOC, PGK1, ALDOA) to regulate the glucose metabolic reprogramming of PCa cells. Conclusion: PCa cells are able to“domesticate”CAFs and directly act on key PCa glycolytic molecules through the release of the exosomal MALAT1 from CAFs, thereby regulating glucose metabolic reprogramming and accelerating PCa development.